The present invention relates to a method for detecting or quantitating an analyte, and to a biosensor for carrying out the method.
Many tools used for detecting or quantitating biological analytes are based on analyte-specific binding between an analyte and an analyte-binding receptor or agent. Analyte/analyte binding pairs encountered commonly in diagnostics include antigen-antibody, hormone-receptor, drug-receptor, cell surface antigen-lectin, biotin-avidin, and complementary nucleic acid strands.
A variety of methods for detecting analyte-binding agent interactions have been developed. The simplest of these is a solid-phase format employing a reporter labeled analyte-binding agent whose binding to or release from a solid surface is dependent on the presence of analyte. In a typical solid-phase sandwich type assay, for example, the analyte to be measured is an analyte with two or more binding sites, allowing analyte binding both to a receptor, e.g., antibody, carried on a solid surface, and to a reporter-labeled second receptor. The presence of analyte is detected (or quantitated) by the presence (or amount) of reporter bound to solid surface.
In a typical solid-phase competitive binding analyte analog for binding to a receptor (analyte-binding agent) carried on a solid support. The amount of reporter signal associated with the solid support is inversely proportional to the amount of sample analyte to be detected or determined.
The reporter label used in both solid-phase formats is typically a visibly detectable particle or an enzyme capable of converting a substrate to an easily detectable product. Simple spectrophotometric devices allow for the quantitation of the amount of reporter label, for quantifying amount of analyte.
Detecting or quantitating analyte-specific binding events is also important in high-throughput methods being developed for combinatorial library screening. In a typical method, a large library of possible effector molecules (analytes) is synthesized. The library members are then screened for effector activity by their ability to bind to a selected receptor. The approach has the potential to identify, for example, new oligopeptide antigens capable of high-specificity binding to disease related antibodies, or small-molecule compounds capable of interacting with a selected pharmacological target, such as a membrane bound receptor or cellular enzyme.
High-throughput screening methods typically employ simple analyte displacement assays to detect and quantitate analyte binding to a receptor. Displacement assays have the advantage of high sensitivity, e.g., where the displaced analyte is radiolabeled, and also allow for the determination of analyte-receptor binding affinity, based on competitive displacement of a binding agent whose binding affinity to the target receptor is known.
In both diagnostics and high-throughput screening, there is increasing interest in developing biosensors capable of detecting and quantifying analyte-receptor binding events.
One general type of biosensor employs an electrode surface in combination with current or impedance measuring elements for detecting a change in current or impedance in response to the presence of a ligand-receptor binding event. This type of biosensor is disclosed, for example, in U.S. Pat. No. 5,567,301.
Gravimetric biosensors employ a piezoelectric crystal to generate a surface acoustic wave whose frequency, wavelength and/or resonance state are sensitive to surface mass on the crystal surface. The shift in acoustic wave properties is therefore indicative of a change in surface mass, e.g., due to a ligand-receptor binding event. U.S. Pat. Nos. 5,478,756 and 4,789,804 describe gravimetric biosensors of this type.
Biosensors based on surface plasmon resonance (SPR) effects have also been proposed, for example, In U.S. Pat. No. 5,485,277. These devices exploit the shift in SPR surface reflection angle that occurs with perturbations, e.g., binding events, at the SPR interface. Finally, a variety of biosensors that utilize changes in optical properties at a biosensor surface are known, e.g., U.S. Pat. No. 5,268,305.
The interest in biosensors is spurred by a number of potential advantages over strictly biochemical assay formats. First, biosensors may be produced, using conventional microchip technology, in highly reproducible and miniaturized form, with the capability of placing a large number of biosensor elements on a single substrate (e.g., see U.S. Pat. Nos. 5,200,051 and 5,212,050).
Secondly, because small signals can be readily amplified (and subjected to various types of signal processing if desired), biosensors have the potential for measuring minute quantities of analyte, and proportionately small changes in analyte levels.
A consequence of the features above is that a large number of different analytes can be detected or quantitated by applying a small sample volume, e.g., 10-50 xcexcl, to a single multi-sensor chip.
Heretofore, electrochemical biosensors have been more successfully applied to detecting analytes that are themselves electrochemical species, or can participate in catalytic reactions that generate electrochemical species, than to detecting analyte-receptor binding events. This is not surprising, given the more difficult challenge of converting a biochemical binding event to an electrochemical signal. One approach to this problem is to provide two separate reaction elements in the biosensor: a first element contains a receptor and bound enzyme-linked analyte, and the second element, components for enzymatically generating and then measuring an electrochemical species. In operation, analyte displaces the analyte-enzyme conjugate from the first element, releasing the enzyme into the second element region, thus generating an electrochemical species which is measured in the second element.
Two-element biosensors of this type are relatively complicated to produce, particularly by conventional silicon-wafer methods, since one or more biological layers and permselective layers must be deposited as part of the manufacturing process. Further, enzymes or receptors in the biosensor can denature on storage, and the device may have variable xe2x80x9cwettingxe2x80x9d periods after a sample is applied.
Biosensors that attempt to couple electrochemical activity directly to an analyte-receptor binding event, by means of gated membrane electrodes, have been proposed. For example, U.S. Pat. Nos. 5,204,239 and 5,368,712 disclose gated membrane electrodes formed of a lipid bilayer membrane containing an ion-channel receptor that is either opened or closed by analyte binding to the receptor. Electrodes of this type are difficult to make and store, and are limited at present to a rather small group of receptor proteins.
Alternatively, direct analyte/receptor binding may be measured electrically by embedding the receptor in a thin polymer film, and measuring changes in the film""s electrical properties, e.g., impedance, due to analyte binding to the receptors. U.S. Pat. No. 5,192,507 is exemplary. Since analyte binding to the receptor will have a rather small effect on film properties, and since no amplification effect is achieved, the approach is expected to have limited sensitivity.
PCT patent application PCT/CA97/00275, published Nov. 6, 1997, publication No. WO 97/41424, discloses a novel electrochemical biosensor having a conductive detection surface, and a hydrocarbon-chain monolayer formed on the surface. Biosensor operation is based on the flow of an ionized redox species across the monolayer, producing a measurable current flow. In one embodiment of the biosensor disclosed, binding of an analyte to its opposite binding member attached to the surface of some of the hydrocarbon chains increases measured current flow by increasing the disorder of the monolayer, making it more permeable to the redox species. In another general embodiment, the opposite binding member is anchored to the monolayer through a coiled-coil heterodimer structure, allowing any selected binding member carried on one (xcex1-helical peptide to be readily attached to a xe2x80x9cuniversalxe2x80x9d monolayer surface carrying the opposite xcex1-helical peptide.
The previously disclosed biosensor is capable of detecting and quantifying analyte-binding events and characterized by: (i) direct electrochemical conversion of the binding event to electrical signal; (ii) a high electron flow xe2x80x9cturnoverxe2x80x9d from each binding event; (iii) adaptable to substantially any analyte, and (iv) good storage characteristics and rapid wetting with sample application.
Given these features, it would be desirable to improve the operational characteristics and suitability of the biosensor to a wide variety of analytes, as well as the adaptability of the biosensor to multianalyte formats, e.g., in a microfabricated form. The present invention is designed to provide these advantages.
The invention includes, in one aspect, a method for detecting or quantitating an analyte present in a liquid sample. The method includes reacting the liquid sample with an analyte-reaction reagent, thereby generating a solution form of a first coil-forming peptide having a selected charge and being capable of interacting with a second, oppositely charged coil-forming peptide to form a stable xcex1-helical coiled-coil heterodimer.
The coil-forming peptide is contacted with a biosensor having a detection surface with surface-bound molecules of such second, oppositely charged coil-forming peptide, under conditions effective to form a stable xcex1-helical coiled-coil heterodimer on the detection surface, where the binding of the solution form of the coil-forming peptide to the immobilized coil-forming peptide is effective to measurably alter a signal generated by the biosensor, which is measured to determine whether such coiled-coil heterodimer formation on said detector surface has occurred.
In one embodiment, the analyte is a ligand, and the reacting includes mixing the analyte with a conjugate of the first coil-forming peptide and the analyte or an analyte analog, and reacting the analyte and conjugate with an analyte-binding anti-ligand agent, such that the amount of unbound conjugate generated is inversely proportional to the amount of analyte. The analyte-bound agent is preferably immobilized. In another related embodiment, the conjugate is bound to the analyte-binding agent, and displaced from the binding agent in the presence of analyte.
In still another embodiment, the analyte is an enzyme and the reacting step is effective to enzymatically release the first coil-forming peptide in soluble form in the presence of analyte.
In one general embodiment, the biosensor is an electrochemical biosensor that includes a conductive detection surface, a monolayer composed of hydrocarbon chains anchored at their proximal ends to the detection surface, and the second charged coil-forming peptide also anchored to said surface, where the binding of the first peptide to the second peptide, to form such heterodimer, is effective to measurably alter current flow across the monolayer mediated by a redox ion species in an aqueous solution in contact with the monolayer, relative to electron flow observed in the presence of the second peptide alone.
Where the redox ion species has the same charge as said second coil-forming peptide, the binding of the first peptide to the second peptide is effective to enhance ion-mediated current flow across said monolayer. Where the redox ion species has a charge opposite that of said second coil-forming peptide, the binding of the first peptide to the second peptide is effective to reduce ion-mediated current flow across said monolayer.
In another aspect, the invention includes a diagnostic device for detecting or quantitating an analyte present in a liquid sample. The device includes a reaction reagent effective to react with analyte to generate a solution form of a first coil-forming peptide having a selected charge and being capable of interacting with a second, oppositely charged coil-forming peptide to form a stable xcex1-helical coiled-coil heterodimer.
A biosensor in the device has a detection surface with surface-bound molecules of a second charged, coil-forming peptide capable of interacting with the first oppositely charged coil-forming peptide to form a stable xcex1-helical coiled-coil heterodimer, where the binding of the first peptide to the second peptide, to form such heterodimer, is effective to measurably alter a signal generated by the biosensor, which is measured by a detector in the device.
The device may further include a substrate having formed therein (i) a sample-introduction region, (ii) the biosensor, and (iii) a sample-flow pathway between said sample-introduction region and the biosensor. The reaction reagent is disposed in the sample-flow pathway and includes a conjugate of the first coil-forming peptide and the analyte or an analyte analog, in a form releasable into the sample liquid, and an analyte-binding agent. The sample-flow pathway may include a mixing zone containing the conjugate in releasable form, and a reaction zone containing the analyte-binding agent in immobilized form.
The device also preferably includes a background control biosensor, and a control sample-flow pathway connecting the sample-introduction region to the background control biosensor, for measuring xe2x80x9cbaselinexe2x80x9d current. The control sample-flow pathway lacks the analyte-conjugate.
In one general embodiment, the biosensor includes a conductive detection surface, a monolayer composed of hydrocarbon chains anchored at their proximal ends to the detection surface, and the second charged coil-forming peptide also anchored to the surface, where the binding of the first peptide to the second peptide, to form such heterodimer, is effective to measurably alter current flow across the monolayer mediated by a redox ion species in an aqueous solution in contact with the monolayer, relative to electron flow observed in the presence of the second peptide alone.
The redox ion species may have the same charge as the second coil-forming peptide, where the binding of the first peptide to the second peptide is effective to enhance redox ion-mediated current flow across the monolayer. Examples are the redox ion species is Fe(CN)63xe2x88x92, if the charge of the second coil-forming peptide is negative, and Ru(NH3)63+, if the charge of the second coil-forming peptide is positive.
Alternatively, the redox ion species may have a charge opposite to that of the second coil-forming peptide, where the binding of the first peptide to the second peptide is effective to reduce ion-mediated current flow across said monolayer. Examples are Fe(CN)63+, if the charge of the second coil-forming peptide is positive, and Ru(NH3)63+, if the charge of said second coil-forming peptide is negative.
In this general embodiment the electrode may have a gold detection surface and the monolayer may be composed of 8-22 carbon atom chains attached at their proximal ends to the detection surface by a thiol linkage, at a molecular density of about 3 to 5 chains/nm2.
The device may be designed for use in detecting or quantitating a plurality of different selected analytes. Here the device includes, for each analyte, (i) a separate biosensor, and (ii) a separate sample-flow pathway connecting the sample-introduction region to each associated biosensor, where each sample-flow pathway includes (i) a conjugate of the first coil-forming peptide and one of the selected analytes or analog thereof, and (ii) an associated selected analyte-binding agent.
Preferably each biosensor in this embodiment contains substantially the same second charged, coil-forming peptide, and the sample-introduction region is a single port communicating with each of the sample-flow pathways. The sample introduction region, biosensors, and sample-flow pathways may be microfabricated on the substrate.
These and other objects and features of the invention will become more fully apparent when the following detailed description of the invention is read in conjunction with the accompanying drawings.